The initiation of protein synthesis is a key step in the gene expression pathway, requiring recruitment and correct positioning of ribosomes on mRNA templates in a process that is highly regulated in all cells. A subset of eukaryotic genes and viruses circumvent the usual cellular controls on initiation by utilizing structured RNAs called internal ribosome entry sites (IRESs) to direct ribosomes to the translational start codon. This project aims to elucidate the mechanism of internal translation initiation by focusing on the IRES elements of Hepatitis C virus (HCV), a major human pathogen, and the cellular c-myc gene, involved in cell growth and transformation. X-ray crystallography will be used to solve molecular structures of functional domains as well as full-length constructs of the HCV IRES RNA, providing a structural basis for exploring IRES interactions with the translational apparatus. Using both cryo-electron microscopy and X-ray crystallography, complexes of the IRES bound to 40S subunits with and without initiation factors will be analyzed. The cryo-EM work will be carried out in an ongoing collaboration with Prof. Joachim Frank, and will guide preparation and crystallization of functional complexes. Structures determined in this part of the study will provide the basis for addressing how the IRES positions the mRNA correctly in the ribosomal decoding center, and how initiation factors influence this assembly process. They will also provide the first images of the human ribosome, enabling comparison with crystallographic and cryo-EM structures of the bacterial, archaeal and yeast ribosomes and subunits. To compare the structural and functional properties of a viral versus a cellular IRES, we will investigate the folding and mechanism of action of the cellular c-myc IRES. Results from this work will yield a detailed mechanistic understanding of one of the fundamental processes in gene expression, and may lead to methods for inhibition of IRES activity.